Phosphodiesesterase 4 inhibitors for the treatment of a cognitive deficit

ABSTRACT

The present invention provides methods of treating cognitive deficits associated with mental retardation. The methods comprise combining cognitive training protocols and a general administration of phosphodiesterase 4 inhibitors.

RELATED APPLICATIONS

This application, which claims benefit under 35 U.S.C. §§120 and 119(e),is a continuation of U.S. application Ser. No. 11/246,005, filed Oct. 7,2005 now U.S. Pat. No. 7,868,015, which is a continuation ofInternational Application No. PCT/US2004/010876, which designated theUnited States and was filed Apr. 8, 2004, published in English, which isa continuation-in-part of U.S. application Ser. No. 10/410,508, filedApr. 8, 2003, now abandoned. The entire teachings of these applicationsare incorporated herein by reference.

BACKGROUND OF THE INVENTION

An estimated 4 to 5 million Americans (about 2% of all ages and 15% ofthose older than age 65) have some form and degree of cognitive failure.Cognitive failure (dysfunction or loss of cognitive functions, theprocess by which knowledge is acquired, retained and used) commonlyoccurs in association with central nervous system (CNS) disorders orconditions, including age-associated memory impairment, delirium(sometimes called acute confusional state), dementia (sometimesclassified as Alzheimer's or non-Alzheimer's type), Alzheimer's disease,Parkinson's disease, Huntington's disease (chorea), mental retardation,cerebrovascular disease (e.g., stroke, ischemia), affective disorders(e.g., depression), psychotic disorders (e.g., schizophrenia, autism(Kanner's Syndrome)), neurotic disorders (e.g., anxiety,obsessive-compulsive disorder), attention deficit disorder (ADD),subdural hematoma, normal-pressure hydrocephalus, brain tumor, head orbrain trauma.

Cognitive dysfunction is typically manifested by one or more cognitivedeficits, which include memory impairment (impaired ability to learn newinformation or to recall previously learned information), aphasia(language/speech disturbance), apraxia (impaired ability to carry outmotor activities despite intact motor function), agnosia (failure torecognize or identify objects despite intact sensory function),disturbance in executive functioning (i.e., planning, organizing,sequencing, abstracting).

Cognitive dysfunction causes significant impairment of social and/oroccupational functioning, which can interfere with the ability of anindividual to perform activities of daily living and greatly impact theautonomy and quality of life of the individual.

Cognitive training protocols are generally employed in rehabilitatingindividuals who have some form and degree of cognitive dysfunction. Forexample, cognitive training protocols are commonly employed in strokerehabilitation and in age-related memory loss rehabilitation. Becausemultiple training sessions are often required before an improvement orenhancement of a specific aspect of cognitive performance (ability orfunction) is obtained in the individuals, cognitive training protocolsare often very costly and time-consuming.

SUMMARY OF THE INVENTION

The present invention relates to a novel methodology, also referred toherein as augmented cognitive training (ACT), which can either (1)rehabilitate various forms of cognitive dysfunction more efficientlythan any current method or (2) enhance normal cognitive performance(ability or function). ACT can be applied for any aspect of brainfunction that shows a lasting performance gain after cognitive training.Accordingly, ACT can be used in rehabilitating an animal with some formand degree of cognitive dysfunction or in enhancing (improving) normalcognitive performance in an animal. ACT can also be used to exerciseappropriate neuronal circuits to fine-tune the synaptic connections ofnewly acquired, transplanted stem cells that differentiate into neurons.

As described herein, ACT comprises two indivisible parts: (1) a specifictraining protocol for each brain (cognitive) function and (2)administration of cyclic AMP response element binding protein (CREB)pathway-enhancing drugs. This combination can augment cognitive trainingby reducing the number of training sessions required to yield aperformance gain relative to that obtained with cognitive training aloneor by requiring shorter or no rest intervals between training sessionsto yield a performance gain. This combination can also augment cognitivetraining by reducing the duration and/or number of training sessionsrequired for the induction in a specific neuronal circuit(s) of apattern of neuronal activity or by reducing the duration and/or numberof training sessions or underlying pattern of neuronal activity requiredto induce CREB-dependent long-term structural/function (i.e.,long-lasting) change among synaptic connections of the neuronal circuit.In this manner, ACT can improve the efficiency of existing cognitivetraining protocols, thereby yielding significant economic benefit.

For example, cognitive training protocols are employed in treatingpatients with depression (monopolor) and/or phobias to help them unlearnpathological responses associated with the depression and/or phobia(s)and learn appropriate behavior. Administration of a CREBpathway-enhancing drug in conjunction with cognitive training reducesthe time and/or number of training sessions required to yield a gain inperformance in these patients. As such, overall treatment isaccomplished in a shorter period of time.

Similarly, cognitive training protocols are employed in treatingpatients with autism to help them unlearn pathological responses and tolearn appropriate behavior. Administration of a CREB pathway-enhancingdrug in conjunction with cognitive training reduces the time and/ornumber of training sessions required to yield a gain in performance inthese patients.

Cognitive training protocols (e.g., physical therapy, bio-feedbackmethods) are employed in rehabilitating stroke patients (strokerehabilitation), particularly rehabilitating impaired or lostsensory-motor function(s). Administration of a CREB pathway-enhancingdrug in conjunction with cognitive training reduces the time and/ornumber of training sessions required to yield a gain in performance inthese patients. Faster and more efficient recovery of lost cognitivefunction(s) are expected as a result.

Cognitive training protocols (e.g., massed training, spaced training)are employed in learning a new language or in learning to play a newmusical instrument. Administration of a CREB pathway-enhancing drug inconjunction with cognitive training reduces the time and/or number oftraining sessions required to yield a gain in performance. As a result,less practice (training sessions) is required to learn the new languageor to learn to play the new musical instrument.

Cognitive training protocols are employed in improving learning and/orperformance in individuals with learning, language or readingdisabilities. Administration of a CREB pathway-enhancing drug inconjunction with cognitive training reduces the time and/or number oftraining sessions required to yield a gain in performance in theseindividuals.

Cognitive training protocols are employed to exercise neuronal circuitsin individuals to fine-tune synaptic connections of newly acquired,transplanted stem cells that differentiate into neurons. Administrationof a CREB pathway-enhancing drug in conjunction with cognitive trainingreduces the time and/or number of training sessions required for theinduction in (a) specific neuronal circuit(s) of a pattern of neuronalactivity in these individuals.

Cognitive training protocols are employed for repeated stimulation ofneuronal activity or a pattern of neuronal activity underlying (a)specific neuronal circuit(s) in individuals. Administration of a CREBpathway-enhancing drug in conjunction with cognitive training reducesthe time and/or number of training sessions and/or underlying pattern ofneuronal activity required to induce CREB-dependent long-termstructure/function (i.e., long-lasting) change among synapticconnections of the neuronal circuit.

As a result of the present invention, methods of enhancing a specificaspect of cognitive performance in an animal (particularly a human orother mammal or vertebrate) in need thereof are provided hereincomprising (a) administering to the animal an augmenting agent whichenhances CREB pathway function; and (b) training the animal underconditions sufficient to produce an improvement in performance of acognitive task of interest by the animal. “Augmenting agents” are alsoreferred to herein as “CREB pathway-enhancing drugs”.

Methods are provided herein for treating a cognitive deficit associatedwith a central nervous system (CNS) disorder or condition in an animalin need of said treatment comprising (a) administering to the animal anaugmenting agent which enhances CREB pathway function; and (b) trainingthe animal under conditions sufficient to produce an improvement inperformance of a particular cognitive task by the animal. CNS disordersand conditions include age-associated memory impairment,neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson'sdisease, Huntington's disease (chorea), other senile dementia),psychiatric diseases (e.g., depression, schizophrenia, autism, attentiondeficit disorder), trauma dependent loss of function (e.g.,cerebrovascular diseases (e.g., stroke, ischemia), brain tumor, head orbrain injury), genetic defects (e.g., Rubinstein-Taybi syndrome, downsyndrome, Angelman syndrome, neurofibromatosis, Coffin-Lowry syndrome,Rett syndrome, myotonic dystrophy, fragile X syndrome (e.g., fragileX-1, fragile X-2), William's syndrome) and learning disabilities.

In a particular embodiment, methods are provided herein for treating acognitive deficit associated with mental retardation in an animal inneed of said treatment comprising (a) administering to the animal anaugmenting agent which enhances CREB pathway function (e.g., aphosphodiesterase 4 inhibitor); and (b) training the animal underconditions sufficient to produce an improvement in performance by theanimal of a cognitive task whose deficit is associated with mentalretardation. The present invention encompasses the use of an augmentingagent which enhances CREB pathway function (e.g., a phosphodiesterase 4inhibitor) for the manufacture of a medicament for use in treatment of acognitive deficit associated with mental retardation. Mental retardationimpacts cognitive processing and cognitive functions, including learningand memory acquisition. Mental retardation may be caused by chromosomalor genetic factors, congenital infections, teratogens (drugs and otherchemicals), malnutrition, radiation or unknown conditions affectingimplantation and embryogenesis. Mental retardation syndromes includeRubinstein-Taybi syndrome, down syndrome, Angelman syndrome,neurofibromatosis, Coffin-Lowry syndrome, Rett syndrome, myotonicdystrophy, fragile X syndrome (e.g., fragile X-1, fragile X-2) andWilliam's syndrome (Weeber, E. J. et al., Neuron, 33:845-848 (2002)). Ina particular embodiment, a phosphodiesterase 4 inhibitor is administeredfor treatment of a cognitive deficit associated with mental retardationat a dose of from about 0.05 to about 20.0 milligrams per kilogram ofbody weight, and preferably at a dose of from about 0.1 to about 10.0milligrams per kilogram of body weight, per administration. In humans,in a particular embodiment, the phosphodiesterase 4 inhibitor isadministered for treatment of a cognitive deficit associated with mentalretardation at a total dose of from about 3.5 to 1,400 milligrams, andpreferably at a total dose of from about 7 to about 700 milligrams, peradministration.

Methods are also provided herein for therapy of a cognitive deficitassociated with a CNS disorder or condition in an animal havingundergone neuronal stem cell manipulation comprising (a) administeringto the animal an augmenting agent which enhances CREB pathway function;and (b) training the animal under conditions sufficient to stimulate orinduce neuronal activity or a pattern of neuronal activity in theanimal. By “neuronal stem cell manipulation” is meant that (1) exogenousneuronal stem cells are transplanted into the brain or spinal chord ofan animal or (2) endogenous neuronal stem cells are stimulated orinduced to proliferate in the animal.

Methods are provided herein for repeated stimulation of neuronalactivity or a pattern of neuronal activity, such as that underlying aspecific neuronal circuit(s), in an animal comprising (a) administeringto the animal an augmenting agent which enhances CREB pathway function;and (b) training the animal under conditions sufficient to stimulate orinduce neuronal activity or a pattern of neuronal activity in theanimal.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating a neuronal mechanism of brainplasticity, which forms the neurological basis for augmented cognitivetraining. Specific cognitive training protocols produce(experience-dependent) changes in neural activity of specific underlyingneuronal circuits. This neural activity activates a biochemical processthat modulates CREB-dependent gene expression. Downstream effectors ofthis transcription factor cascade then yield long-lasting structural andfunctional changes in synaptic connectivity of the circuit (i.e.,long-term memory) (Dubnau J. et al., Current Biology, 13: 286-296(2003)). This process of experience-dependent synaptic modification isongoing in normal animals and usually requires multiple trainingsessions for most tasks. Augmentation of the CREB pathway duringtraining will reduce the number of training sessions (or shorten therest interval between them) required to produce the experience-dependentchanges in synaptic structure and/or function.

FIG. 2A is a bar graph representation depicting results showing that thePDE4 inhibitors rolipram and HT0712 enhance forskolin-induced geneexpression in human neuroblastoma cells. Relative light units (RLU)emitted from luciferase were quantified in human neuroblastoma cellsstably transfected with a CRE-luciferase reporter gene and exposed tovehicle alone or drug (HT0712 or rolipram) for two hours beforestimulation by a suboptimal dose of forskolin. The results show thatboth drugs increased forskolin-induced CRE-luciferase expression1.9-fold above forskolin alone when assayed 4 hours after forskolinstimulation.

FIG. 2B is a bar graph representation depicting results showing thatthe. PDE4 inhibitors rolipram and HT0712 enhance forskolin-induced geneexpression in human neuroblastoma cells. Real-time PCR was used toquantify expression of somatostatin, an endogenous cAMP-responsive gene.Expression levels induced by forskolin or by forskolin+drug arequantified as differences in threshold cycle number (AC) above vehiclealone control groups. The results show that HT0712 and rolipram produced4.6-fold and 2.3-fold increases in forskolin-induced expression ofsomatostatin, respectively.

FIG. 3 is a bar graph representation depicting results showing thatCBP^(+/−) mice have impaired long term memory in object recognitiontask. Wildtype mice and CBP^(+/−) mutant mice were trained for 15minutes and tested 3 hours or 24 hours later. Memory retention wasquantified as a Discrimination Index, the fraction of time spentexploring a novel versus a familiar object. Three-hour memory levelswere similar for normal mice and CBP^(+/−) mutants (p=0.76; n=6 for eachgenotype), but 24 hour memory was significantly lower than normal inmutant mice (p<0.01; n=10 for each genotype).

FIG. 4 is a bar graph representation depicting results showing that thePDE4 inhibitors HT0712 and rolipram ameliorate the long-term memorydefect in CBP^(+/−) mutant mice. Wildtype mice and CBP^(+/−) mutant micereceived 0.1 mg/kg HT 0712 or rolipram injected i.p. 20 minutes beforetraining. Animals experienced a 15 minute training session and memoryretention was tested 24 hours later. In vehicle-injected CBP^(+/−)mutants, memory was significantly lower than in vehicle-injectedwildtype mice (p<0.01; n=12 and n=6, respectively). In drug-injectedCBP^(+/−) mutants, memory was significantly higher than invehicle-injected mutants (p<0.05; N=10 and N=12, respectively, forHT0712; p<0.05, N=8 and N=2, respectively, for rolipram). Memoryretention did not differ significantly between drug-treated CBP^(+/−)mutants and drug-treated wildtype mice (p=0.78, N=10 for each group forHT0712; p=0.19, N=8 and N=10, respectively, for rolipram).

FIG. 5 is a bar graph representation depicting results showing a doseresponse curve for HT0712 in wildtype mice. Mice received a single i.p.injection of drug or vehicle alone 20 minutes before training. Doses of0.001 mg/kg, 0.005 mg/kg, 0.01 mg/kg, 0.05 mg/kg, 0.10 mg/kg, 0.15mg/kg, 0.20 mg/kg and 0.50 mg/kg were used. Animals experienced a 3.5minute training protocol and were tested 24 hours later. Memoryretention in drug-injected animals was significantly higher than that invehicle-alone animals (N=35) at doses of 0.05 mg/kg (N=20; p<0.05), 0.10mg/kg (N=22; p<0.0001) and 0.15 mg/kg (N=18; p<0.001).

FIG. 6 is a graphical representation depicting results showing thatCBP^(+/−) mutants and wildtype mice show a different dose sensitivity toHT0712. CBP^(+/−) mutants and wildtype mice received a single i.p.injection of vehicle or drug 20 minutes before training. Theyexperienced a 3.5 minute training protocol and were tested 24 hourslater. In wildtype mice, memory retention in drug-treated groups washigher than in the vehicle-alone group (N=26) at doses of 0.05 mg/kg(N=12; p<0.005), 0.10 mg/kg (N=8; p<0.0001), 0.15 mg/kg (N=18; p<0.005)and 0.2 mg/kg (N=14; p<0.005). In CBP^(+/−) mutants, memory retention indrug-treated groups was higher than in the vehicle along group (N=26) atdoses of 0.10 mg/kg (N=8; p<0.0001), 0.15 mg/kg (N=10; p<0.0001) and 0.2mg/kg (N=14; p<0.0001). In contrast to wildtype mice, a 0.05 mg/kg doseof HT0712 failed to enhance memory in CBP^(+/−) mutants (N=26; p=0.79).

DETAILED DESCRIPTION OF THE INVENTION

For many tasks in many species, including human, spaced trainingprotocols (multiple training sessions with a rest interval between each)produce stronger, longer-lasting memory than massed training protocols(multiple training sessions with no rest interval in between).Behavior-genetic studies of Pavlovian olfactory learning in Drosophilahave established that massed training produces a long-lasting memorythat nevertheless decays away in at least four days, is not proteinsynthesis-dependent, is not disrupted by overexpression of aCREB-repressor transgene, and is disrupted in radish mutants (Tully, T.et al., Cell, 79(1):35-47 (1994); and Yin, J. C. et al., Cell,79(1):49-58 (1994)). In contrast, spaced training produces along-lasting memory that persists for at least seven days, is proteinsynthesis-dependent, is disrupted by overexpression of a CREB-repressortransgene and is normal in radish mutants (Tully, T. et al., Cell,79(1):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)).One day after spaced training, memory retention is composed of both theprotein synthesis- and CREB-independent early memory (ARM) and theprotein synthesis- and CREB-dependent long-term memory (LTM). Additionalmassed training is insufficient to induce LTM (Tully, T. et al., Cell,79(1):35-47 (1994); and Yin, J. C. et al., Cell, 79(1):49-58 (1994)).

A growing body of evidence extends these results from invertebrates tomammals. For example, in Aplysia, molecular manipulations of CREBexpression, similar to those in flies, suppress or enhance (i) LTM of afacilitatory electrophysiological response at a sensorimotor monosynapsein cell culture and (ii) the synaptic connections between sensory andmotor neurons that are normally produced after spaced applications ofthe facilitatory stimulus (Bartsch, D. et al., Cell, 83(6):979-992(1995)). In rats, injections of antisense RNA oligonucleotides intohippocampus or amygdala block LTM formation of two different tasks thatare dependent on activity in these anatomical regions, respectively(Guzowski, J. F. et al., Proc. Natl. Acad. Sci. USA, 94(6):2693-2698(1997); and Lamprecht, R. et al., J. Neurosci., 17(21):8443-8450(1997)). In mice, LTM formation for both implicit and explicit tasks isdefective in CREB mutant mice (Bourtchuladze, R. et al., Cell,79(1):59-68 (1994)).

Training of transgenic mice, carrying a CRE-dependent reporter gene(beta-galactosidase), in hippocampal-dependent contextual fearconditioning or passive avoidance tasks induces CRE-dependent reportergene expression in areas CA1 and CA3 of the hippocampus. Training ofthese mice in an amygdala-dependent fear conditioning task inducesCRE-dependent reporter gene expression in the amygdala, but not thehippocampus. Thus, training protocols that induce LTM formation alsoinduce CRE-dependent gene transcription in specific anatomical areas ofthe mammalian brain (Impey, S. et al., Nat. Neurosci., 1(7):595-601(1998)).

With these animal models, three salient cases of LTM enhancement havebeen demonstrated. First, overexpression of a CREB-activator transgeneabrogates the requirements for multiple, spaced training sessions and,instead, induces LTM formation after only one training session (whichnormally produces little or no memory retention 24 hours later (Yin, J.C. et al., Cell, 81(1):107-115 (1995)). Second, injection of a virallyexpressed CREB-activator transgene into rat amygdala also is sufficientto enhance memory after massed training for the fear-potentiated startleresponse, which abrogates the requirement for a rest interval in spacedtraining (Josselyn, S. A. et al., Society for Neuroscience, Vol. 24,Abstract 365.10 (1998); and Josselyn, S. A. et al., J. Neurosci.,21:2404-2412 (2001)). Third, LTM formation in CREB-deficient mice(Bourtchuladze, R. et al., Cell, 79(1):59-68 (1994)) can form normally,if mutant mice are subjected to a different, spaced training protocol(Kogan, J. H. et al., Curr. Biol., 7(1):1-11 (1997)).

CREB also appears involved in various forms of developmental andcellular plasticity in the vertebrate brain. For example, neuronalactivity increases CREB activity in the cortex (Moore, A. N. et al., J.Biol. Chem., 271(24):14214-14220 (1996)). CREB also mediatesdevelopmental plasticity in the hippocampus (Murphy, D. D. et al., Proc.Natl. Acad. Sci. USA, 94(4):1482-1487 (1997)), in the somatosensorycortex (Glazewski, S. et al., Cereb. Cortex, 9(3):249-256 (1999)), inthe striatum (Liu, F. C. et al., Neuron, 17(6):1133-1144 (1996)), and inthe visual cortex (Pham, T. A. et al., Neuron, 22(1):63-72 (1999)).

CREB appears to be affected in human neurodegenerative disease and braininjury. For example, CREB activation and/or expression is disrupted inAlzheimer's disease (Ikezu, T. et al., EMBO J., 15(10):2468-2475 (1996);Sato, N. et al., Biochem. Biophys. Res. Commun., 232(3):637-642 (1997);Yamamoto-Sasaki, M. et al., Brain. Res., 824(2):300-303 (1999); Vitolo,O. V. et al., Proc. Natl. Acad. Sci. USA, 13217-13221 (2002)). CREBactivation and/or expression is also elevated after seizures or ischemia(Blendy, J. A. et al., Brain Res., 681(1-2):8-14 (1995); and Tanaka, K.et al., Neuroreport, 10(11):2245-2250 (1999)). “Environmentalenrichment” is neuroprotective, preventing cell death by acting throughCREB (Young, D. et al., Nat. Med., 5(4):448-453 (1999)).

CREB functions during drug sensitivity and withdrawal. For example, CREBis affected by ethanol (Pandey, S. C. et al., Alcohol Clin. Exp. Res.,23(9):1425-1434 (1999); Constantinescu, A. et al., J. Biol. Chem.,274(38):26985-26991 (1999); Yang, X. et al., Alcohol Clin. Exp. Res.,22(2):382-390 (1998); Yang, X. et al., J. Neurochem., 70(1):224-232(1998); and Moore, M. S. et al., Cell, 93(6):997-1007 (1998)), bycocaine (Carlezon, W. A., Jr. et al., Science, 282(5397):2272-2275(1998)), by morphine (Widnell, K. L. et al., J. Pharmacol. Exp. Ther.,276(1):306-315 (1996)), by methamphetamine (Muratake, T. et al., AnnN.Y. Acad. Sci., 844:21-26 (1998)) and by cannabinoid (Calandra, B. etal., Eur. J. Pharmacol., 374(3):445-455 (1999); and Herring, A. C. etal., Biochem. Pharmacol., 55(7):1013-1023 (1998)).

A signal transduction pathway that can stimulate the CREB/CREtranscriptional pathway is the cAMP regulatory system. Consistent withthis, mice lacking both adenylate cyclase 1 (AC1) and AC8 enzymes failto learn (Wong S. T. et al., Neuron, 23(4):787-798 (1999)). In thesemice, administration of forskolin to area CA1 of the hippocampusrestores learning and memory of hippocampal-dependent tasks.Furthermore, treatment of aged rats with drugs that elevate cAMP levels(such as rolipram and D1 receptor agonists) ameliorates an age-dependentloss of hippocampal-dependent memory and cellular long-term potentiation(Barad, M. et al., Proc. Natl. Acad. Sci. USA, 95(25):15020-15025(1998)). These latter data suggest that a cAMP signaling is defective inlearning-impaired aged rats (Bach, M. E. et al., Proc. Natl. Acad. Sci.USA, 96(9):5280-5285 (1999)).

The present invention relates to a novel methodology, also referred toherein as augmented cognitive training (ACT), which can (1) rehabilitatevarious forms of cognitive dysfunction or (2) enhance normal cognitiveperformance. ACT acts via a general molecular mechanism of synapticplasticity, which apparently converts the biochemical effect of a newlyacquired experience into a long-lasting structural change of thesynapse. ACT can be applied for any aspect of brain function that showsa lasting performance gain after cognitive training. Accordingly, ACTcan be used in rehabilitating an animal with any form of cognitivedysfunction or in enhancing or improving any aspect of normal cognitiveperformance in an animal.

A growing body of evidence suggests that neurons continue to proliferatein the adult brain (Arsenijevic, Y. et al., Exp. Neurol., 170: 48-62(2001); Vescovi, A. L. et al., Biomed. Pharmacother., 55:201-205 (2001);Cameron, H. A. and McKay, R. D., J. Comp. Neurol., 435:406-417 (2001);and Geuna, S. et al., Anat. Rec., 265:132-141 (2001)) and that suchproliferation is in response to various experiences (Nilsson, M. et al.,J. Neurobiol., 39:569-578 (1999); Gould, E. et al., Trends Cogn. Sci.,3:186-192 (1999); Fuchs, E. and Gould, E., Eur. J. Neurosci.,12:2211-2214 (2000); Gould, E. et al., Biol. Psychiatry, 48:715-720 (2000);and Gould, E. et al., Nat. Neurosci., 2:260-265 (1999)). Experimentalstrategies now are underway to transplant neuronal stem into adult brainfor various therapeutic indications (Kurimoto, Y. et al., Neurosci.Lett., 306:57-60 (2001); Singh, G., Neuropathology, 21:110-114 (2001);and Cameron, H. A. and McKay, R. D., Nat. Neurosci., 2:894-897 (1999)).Much already is known about neurogenesis in embryonic stages ofdevelopment (Saitoe, M. and Tully, T., “Making connections betweensynaptic and behavioral plasticity in Drosophila”, In Toward a Theory ofNeuroplasticity, J. McEachem and C. Shaw, Eds. (New York: PsychologyPress.), pp. 193-220 (2000)). Neuronal differentiation, neuriteextension and initial synaptic target recognition all appear to occur inan activity-independent fashion. Subsequent synaptogenesis and synapticgrowth, however, then requires ongoing neuronal activity to fine-tunesynaptic connections in a functionally relevant manner. These findingssuggest that functional (final) integration of transplanted neural stemcells require neuronal activity. Thus, ACT can be used to exerciseappropriate neuronal circuits to fine-tune the synaptic connections ofnewly acquired, transplanted stem cells that differentiate into neurons.By “exercise appropriate neuronal circuit(s)” is meant the induction inthe appropriate neuronal circuit(s) of a pattern of neuronal activity,which corresponds to that produced by a particular cognitive trainingprotocol. The cognitive training protocol can be used to induce suchneuronal activity. Alternatively, neuronal activity can be induced bydirect electrical stimulation of the neuronal circuitry. “Neuronalactivity” and “neural activity” are used interchangeably herein.

ACT comprises a specific training protocol for each brain function and ageneral administration of CREB pathway-enhancing drugs. The trainingprotocol (cognitive training) induces neuronal activity in specificbrain regions and produces improved performance of a specific brain(cognitive) function. CREB pathway-enhancing drugs, also referred toherein as augmenting agents, enhance CREB pathway function, which isrequired to consolidate newly acquired information into LTM. By “enhanceCREB pathway function” is meant the ability to enhance or improveCREB-dependent gene expression. CREB-dependent gene expression can beenhanced or improved by increasing endogenous CREB production, forexample by directly or indirectly stimulating the endogenous gene toproduce increased amounts of CREB, or by increasing functional(biologically active) CREB. See, e.g., U.S. Pat. Nos. 5,929,223;6,051,559; and International Publication No. WO9611270 (published Apr.18, 1996), which references are incorporated herein in their entirety byreference. Administration of CREB pathway-enhancing drugs decreases thetraining needed to yield a performance gain relative to that yieldedwith training alone. In particular, ACT can enhance cognitive trainingby reducing the number of training sessions required to yield aperformance gain relative to that yielded with cognitive training aloneor by requiring shorter or no rest intervals between training sessionsto yield a performance gain. In this manner, ACT can improve theefficiency of cognitive training techniques, thereby yieldingsignificant economic benefit. By “performance gain” is meant animprovement in an aspect of cognitive performance.

The invention provides methods for enhancing a specific aspect ofcognitive performance in an animal (particularly in a human or othermammal or vertebrate) in need thereof comprising (a) administering tothe animal an augmenting agent which enhances CREB pathway function; and(b) training the animal under conditions sufficient to produce animprovement in performance of a particular cognitive task by the animal.

Training can comprise one or multiple training sessions and is trainingappropriate to produce an improvement in performance of the cognitivetask of interest. For example, if an improvement in language acquisitionis desired, training would focus on language acquisition. If animprovement in ability to learn to play a musical instrument is desired,training would focus on learning to play the musical instrument. If animprovement in a particular motor skill is desired, training would focuson acquisition of the particular motor skill. The specific cognitivetask of interest is matched with appropriate training.

The invention also provides methods for repeated stimulation of neuronalactivity or a pattern of neuronal activity, such as that underlying aspecific neuronal circuit(s), in an animal comprising (a) administeringto the animal an augmenting agent which enhances CREB pathway function;and (b) training the animal under conditions sufficient to stimulate orinduce neuronal activity or a pattern of neuronal activity in theanimal. In this case, training is training appropriate to stimulate orinduce neuronal activity or a pattern of neuronal activity in theanimal.

By “multiple training sessions” is meant two or more training sessions.The augmenting agent can be administered before, during or after one ormore of the training sessions. In a particular embodiment, theaugmenting agent is administered before and during each trainingsession. Treatment with augmenting agent in connection with eachtraining session is also referred to as the “augmenting treatment”. By“training” is meant cognitive training.

Cognitive training protocols are known and readily available in the art.See for example, Karni, A. and Sagi, D., “Where practice makes perfectin text discrimination: evidence for primary visual cortex plasticity”,Proc. Natl. Acad. Sci. USA, 88:4966-4970 (1991); Karni, A. and Sagi, D.,“The time course of learning a visual skill”, Nature, 365:250-252(1993); Kramer, A. F. et al., “Task coordination and aging: explorationsof executive control processes in the task switching paradigm”, ActaPsychol. (Amst.), 101:339-378 (1999); Kramer, A. F. et al., “Trainingfor executive control: Task coordination strategies and aging”, In Agingand Skilled Performance: Advances In Theory and Applications, W. Rogerset al., eds. (Hillsdale, N.J.: Erlbaum) (1999); Rider, R. A. andAbdulahad, D. T., “Effects of massed versus distributed practice ongross and fine motor proficiency of educable mentally handicappedadolescents”, Percept. Mot. Skills, 73:219-224 (1991); Willis, S. L. andSchaie, K. W., “Training the elderly on the ability factors of spatialorientation and inductive reasoning”, Psychol. Aging, 1:239-247 (1986);Willis, S. L. and Nesselroade, C. S., “Long-term effects of fluidability training in old-old age”, Develop. Psychol., 26:905-910 (1990);Wek, S. R. and Husak, W. S., “Distributed and massed practice effects onmotor performance and learning of autistic children”, Percept. Mot.Skills, 68:107-113 (1989); Verhaehen, P. et al., “Improving memoryperformance in the aged through mnemonic training: a meta-analyticstudy”, Psychol. Aging, 7:242-251 (1992); Verhaeghen, P. and Salthouse,T. A., “Meta-analyses of age-cognition relations in adulthood: estimatesof linear and nonlinear age effects and structural models”, Psychol.Bull., 122:231-249 (1997); Dean, C. M. et al., “Task-related circuittraining improves performance of locomotor tasks in chronic stroke: arandomized, controlled pilot trial”, Arch. Phys. Med. Rehabil.,81:409-417 (2000); Greener, J. et al., “Speech and language therapy foraphasia following stroke”, Cochrane Database Syst. Rev., CD000425(2000); Hummelsheim, H. and Eickhof, C., “Repetitive sensorimotortraining for arm and hand in a patient with locked-in syndrome”, Scand.J. Rehabil. Med., 31:250-256 (1999); Johansson, B. B., “Brain plasticityand stroke rehabilitation. The Willis lecture”, Stroke, 31:223-230(2000); Ko Ko, C., “Effectiveness of rehabilitation for multiplesclerosis”, Clin. Rehabil., 13 (Suppl. 1):33-41 (1999); Lange, G. etal., “Organizational strategy influence on visual memory performanceafter stroke: cortical/subcortical and left/right hemisphere contrasts”,Arch. Phys. Med. Rehabil., 81:89-94 (2000); Liepert, J. et al.,“Treatment-induced cortical reorganization after stroke in humans”,Stroke, 31:1210-1216 (2000); Lotery, A. J. et al., “Correctable visualimpairment in stroke rehabilitation patients”, Age Ageing, 29:221-222(2000); Majid, M. J. et al., “Cognitive rehabilitation for memorydeficits following stroke” (Cochrane review), Cochrane Database Syst.Rev., CD002293 (2000); Merzenich, M. et al., “Cortical plasticityunderlying perceptual, motor, and cognitive skill development:implications for neurorehabilitation”, Cold Spring Harb. Symp. Quant.Biol., 61:1-8 (1996); Merzenich, M. M. et al., “Temporal processingdeficits of language-learning impaired children ameliorated bytraining”, Science, 271:77-81 (1996); Murphy, E., “Strokerehabilitation”, J. R. Coll. Physicians Lond., 33:466-468 (1999);Nagarajan, S. S. et al., “Speech modifications algorithms used fortraining language learning-impaired children”, IEEE Trans. Rehabil.Eng., 6:257-268. (1998); Oddone, E. et al., “Quality EnhancementResearch Initiative in stroke: prevention, treatment, andrehabilitation”, Med. Care 38:192-1104 (2000); Rice-Oxley, M. andTurner-Stokes, L., “Effectiveness of brain injury rehabilitation”, Clin.Rehabil., 13(Suppl 1):7-24 (1999); Tallal, P. et al., “Language learningimpairments: integrating basic science, technology, and remediation”,Exp. Brain Res., 123:210-219 (1998); Tallal, P. et al., “Languagecomprehension in language-learning impaired children improved withacoustically modified speech”, Science, 271:81-84 (1996); Wingfield, A.et al., “Regaining lost time: adult aging and the effect of timerestoration on recall of time-compressed speech”, Psychol. Aging,14:380-389 (1999), which references are incorporated herein in theirentirety by reference.

As used herein, the term “animal” includes mammals, as well as otheranimals, vertebrate and invertebrate (e.g., birds, fish, reptiles,insects (e.g., Drosophila species), mollusks (e.g., Aplysia). The terms“mammal” and “mammalian”, as used herein, refer to any vertebrateanimal, including monotremes, marsupials and placental, that suckletheir young and either give birth to living young (eutharian orplacental mammals) or are egg-laying (metatharian or nonplacentalmammals). Examples of mammalian species include humans and primates(e.g., monkeys, chimpanzees), rodents (e.g., rats, mice, guinea pigs)and ruminents (e.g., cows, pigs, horses).

The animal can be an animal with some form and degree of cognitivedysfunction or an animal with normal cognitive performance (i.e., ananimal without any form of cognitive failure (dysfunction or loss of anycognitive function)).

Cognitive dysfunction, commonly associated with brain dysfunction andcentral nervous system (CNS) disorders or conditions, arises due toheredity, disease, injury and/or age. CNS disorders and conditionsassociated with some form and degree of cognitive failure (dysfunction)include, but are not limited to the following:

1) age-associated memory impairment;

2) neurodegenerative disorders, such as delirium (acute confusionalstate); dementia, including Alzheimer's disease and non-Alzheimer's typedementias, such as, but not limited to, Lewy body dementia, vasculardementia, Binswanger's dementia (subcortical arterioscleroticencephalopathy), dementias associated with Parkinson's disease,progressive supranuclear palsy, Huntington's disease (chorea), Pick'sdisease, normal-pressure hydrocephalus, Creutzfeldt-Jakob disease,Gerstmann-Sträussler-Scheinker disease, neurosyphilis (general paresis)or HIV infection, frontal lobe dementia syndromes, dementias associatedwith head trauma, including dementia pugilistica, brain trauma, subduralhematoma, brain tumor, hypothyroidism, vitamin B₁₂ deficiency,intracranial radiation; other neurodegenerative disorders;

3) psychiatric disorders, including affective disorders (mooddisorders), such as, but not limited to, depression, includingdepressive pseudodementia; psychotic disorders, such as, but not limitedto, schizophrenia and autism (Kanner's Syndrome); neurotic disorders,such as, but not limited to, anxiety and obsessive-compulsive disorder;attention deficit disorder;

4) trauma-dependent loss of cognitive function, such as, but not limitedto that associated with (due to), cerebrovascular diseases, includingstroke and ischemia, including ischemic stroke; brain trauma, includingsubdural hematoma and brain tumor; head injury;

5) disorders associated with some form and degree of cognitivedysfunction arising due to a genetic defect, such as, but not limitedto, Rubinstein-Taybi syndrome, down syndrome, Angelman syndrome, fragileX syndrome (fragile X-1, fragile X-2), neurofibromatosis, Coffin-Lowrysyndrome, myotonic dystrophy, Rett syndrome, William's syndrome,Klinefelter's syndrome, mosaicisms, trisomy 13 (Patau's syndrome),trisomy 18 (Edward's syndrome), Turner's syndrome, cri du chat syndrome,Lesch-Nyhan syndrome (hyperuricemia), Hunter's syndrome, Lowe'soculocerebrorenal syndrome, Gaucher's disease, Hurler's syndrome(mucopolysaccharidosis), Niemann-Pick disease, Tay-Sachs disease,galactosemia, maple syrup urine disease, phenylketonuria,aminoacidurias, acidemias, tuberous sclerosis and primary microcephaly;

6) learning, language or reading disabilities, particularly in children.By “learning disabilities” is meant disorders of the basic psychologicalprocesses that affect the way an individual learns. Learningdisabilities can cause difficulties in listening, thinking, talking,reading, writing, spelling, arithmetic or combinations of any of theforegoing. Learning disabilities include perceptual handicaps, dyslexiaand developmental aphasia.

The terms “cognitive performance” and “cognitive function” areart-recognized terms and are used herein in accordance with theirart-accepted meanings. By “cognitive task” is meant a cognitivefunction. Cognitive functions include memory acquisition, visualdiscrimination, auditory discrimination, executive functioning, motorskill learning, abstract reasoning, spatial ability, speech and languageskills and language acquisition. By “enhance a specific aspect ofcognitive performance” is meant the ability to enhance or improve aspecific cognitive or brain function, such as, for example, theacquisition of memory or the performance of a learned task. By“improvement in performance of a particular cognitive task” is meant animprovement in performance of a specific cognitive task or aspect ofbrain function relative to performance prior to training. For example,if after a stroke, a patient can only wiggle his or her toe, animprovement in performance (performance gain) in the patient would bethe ability to walk, for example.

Accordingly, the invention also relates to methods of treating acognitive deficit associated with a CNS disorder or condition in ananimal (particularly in a human or other mammal or vertebrate) in needof said treatment comprising (a) administering to the animal anaugmenting agent which enhances CREB pathway function; and (b) trainingthe animal under conditions sufficient to produce an improvement inperformance of a particular cognitive task by the animal.

In one embodiment, the invention relates to a method of treating acognitive deficit associated with age-associated memory impairment in ananimal in need of said treatment comprising (a) administering to theanimal an augmenting agent which enhances CREB pathway function; and (b)training the animal under conditions sufficient to produce animprovement in performance by the animal of a cognitive task whose lossis associated with age-associated memory impairment.

In a second embodiment, the invention relates to a method of treating acognitive deficit associated with a neurodegenerative disease (e.g.,Alzheimer's disease, Parkinson's disease, Huntington's disease, othersenile dementia) in an animal in need of said treatment comprising (a)administering to the animal an augmenting agent which enhances CREBpathway function; and (b) training the animal under conditionssufficient to produce an improvement in performance by the animal of acognitive task whose deficit is associated with the neurodegenerativedisease.

In a third embodiment, the invention relates to a method of treating acognitive deficit associated with a psychiatric disease (e.g.,depression, schizophrenia, autism, attention deficit disorder) in ananimal in need of said treatment comprising (a) administering to theanimal an augmenting agent which enhances CREB pathway function; and (b)training the animal under conditions sufficient to produce animprovement in performance by the animal of a cognitive task whosedeficit is associated with the psychiatric disease.

In a fourth embodiment, the invention relates to a method of treating acognitive deficit associated with trauma dependent loss of cognitivefunction (e.g., cerebrovascular diseases (e.g., stroke, ischemia), braintumor, head or brain injury) in an animal in need of said treatmentcomprising (a) administering to the animal an augmenting agent whichenhances CREB pathway function; and (b) training the animal underconditions sufficient to produce an improvement in performance by theanimal of a cognitive task whose deficit is associated with traumadependent loss of cognitive function.

In a fifth embodiment, the invention relates to a method of treating acognitive deficit associated with a genetic defect (e.g.,Rubinstein-Taybi syndrome, down syndrome, Angelman syndrome,neurofibromatosis, Coffin-Lowry syndrome, Rett syndrome, myotonicdystrophy, fragile X syndrome (e.g., fragile X-1, fragile X-2) andWilliam's syndrome) in an animal in need of said treatment comprising(a) administering to the animal an augmenting agent which enhances CREBpathway function; and (b) training the animal under conditionssufficient to produce an improvement in performance by the animal of acognitive task whose deficit is associated with a genetic defect.

In a particular embodiment, the invention relates to methods of treatinga cognitive deficit associated with mental retardation in an animal inneed of said treatment comprising (a) administering to the animal anaugmenting agent which enhances CREB pathway function; and (b) trainingthe animal under conditions sufficient to produce an improvement inperformance by the animal of a cognitive task whose deficit isassociated with mental retardation. The invention encompasses the use ofan augmenting agent which enhances CREB pathway function for manufactureof a medicament for use in treatment of a cognitive deficit associatedwith mental retardation. In a particular embodiment, the augmentingagent is a phosphodiesterase 4 (PDE4) inhibitor. Examples of PDE4inhibitors include rolipram and compounds of the following formula:

wherein “Me” means “methyl” and “cPent” means “cyclopentyl”. It isunderstood that the above formula embraces both enantimers and mixturesthereof. The compounds can be prepared using the methodology provided inU.S. Pat. No. 6,458,829, the teachings of which are incorporated hereinby reference. In a particular embodiment, the 3 and 5 carbons of thisabove formula are in the S configuration:

wherein “Me” means “methyl” and “cPent” means “cyclopentyl”. Otherexamples of PDE4 inhibitors can be found in U.S. Publication No.2002/0028842 A1 (published Mar. 7, 2002); U.S. Pat. No. 6,458,829B1;6,525,055B1; 5,552,438; 6,436,965; and 6,204,275. Still other PDE4inhibitors are known and readily available in the art.

Mental retardation impacts cognitive processing and cognitive functions,including learning and memory acquisition (Weeber, E. J. et al., Neuron,33:845-848)). Mental retardation may be caused by chromosomal or geneticfactors, congenital infections, teratogens (drugs and other chemicals),malnutrition, radiation or unknown conditions affecting implantation andembryogenesis. Mental retardation syndromes include, but are not limitedto, Klinefelter's syndrome, mosaicisms, trisomy 13 (Patau's syndrome),trisomy 18 (Edward's syndrome), Turner's syndrome, cri du chat syndrome,Lesch-Nyhan syndrome (hyperuricemia), Hunter's syndrome, Lowe'soculocerebrorenal syndrome, Gaucher's disease, Hurler's syndrome(mucopolysaccharidosis), Niemann-Pick disease, Tay-Sachs disease,galactosemia, maple syrup urine disease, phenylketonuria,aminoacidurias, acidemias, tuberous sclerosis and primary microcephaly.Mental retardation syndromes also include Rubinstein-Taybi syndrome,down syndrome, Angelman syndrome, neurofibromatosis, Coffin-Lowrysyndrome, Rett syndrome, myotonic dystrophy, fragile X syndrome (e.g.,fragile X-1, fragile X-2) and William's syndrome (Weeber, E. J. et al.,Neuron, 33:845-848 (2002)).

The invention also relates to methods of therapy of a cognitive deficitassociated with a CNS disorder or condition in an animal havingundergone neuronal stem cell manipulation comprising (a) administeringto the animal an augmenting agent which enhances CREB pathway function;and (b) training the animal under conditions sufficient to stimulate orinduce neuronal activity or a pattern of neuronal activity in theanimal. By “neuronal stem cell manipulation” is meant that (1) exogenousneuronal stem cells are transplanted into the brain or spinal chord ofan animal or (2) endogenous neuronal stem cells are stimulated orinduced to proliferate in the animal. Methods of transplanting neuronalstem cells into the brain or spinal chord of an animal are known andreadily available in the art (see, e.g., Cameron, H. A. and McKay, R.D., Nat. Neurosci., 2:894-897 (1999); Kurimoto, Y. et al., Neurosci.Lett., 306:57-60 (2001); and Singh, G., Neuropathology, 21:110-114(2001)). Methods of stimulating or inducing proliferation of endogenousneuronal stem cells in an animal are known and readily available in theart (see, e.g., Gould, E. et al., Trends Cogn. Sci., 3:186-192 (1999);Gould, E. et al., Biol. Psychiatry, 48:715-20 (2000); Nilsson, M. et al,J. Neurobiol., 39:569-578 (1999); Fuchs, E. and Gould, E., Eur. J.Neurosci., 12:2211-2214 (2000); and Gould, E. et al., Nat. Neurosci.,2:260-265 (1999)). The particular methods of transplanting neuronal stemcells into the brain or spinal chord of an animal and the particularmethods of stimulating or inducing proliferation of endogenous neuronalstem cells in an animal are not critical to the practice of theinvention.

The invention further relates to methods of improving or enhancinglearning and/or performance in an animal with a learning, language orreading disability, or combinations of any of the foregoing, comprising(a) administering to the animal an augmenting agent which enhances CREBpathway function; and (b) training the animal under conditionssufficient to produce an improvement in performance by the animal of acognitive task associated with the disability in learning, language orreading performance.

Augmenting agents, as used herein, are compounds with pharmacologicalactivity and include drugs, chemical compounds, ionic compounds, organiccompounds, organic ligands, including cofactors, saccharides,recombinant and synthetic peptides, proteins, peptoids, nucleic acidsequences, including genes, nucleic acid products, and other moleculesand compositions.

For example, augmenting agents can be cell permeant cAMP analogs (e.g,8-bromo cAMP); activators of adenylate cyclase 1 (AC1) (e.g.,forskolin); agents affecting G-protein linked receptor, such as, but notlimited to adrenergic receptors and opioid receptors and their ligands(e.g., phenethylamines); modulators of intracellular calciumconcentration (e.g., thapsigargin, N-methyl-D-aspartate (NMDA) receptoragonists); inhibitors of the phosphodiesterases responsible for cAMPbreakdown (e.g., phosphodiesterase 1 (PDE1) inhibitors (e.g.,iso-buto-metho-xanthine (IBMX)), phosphodiesterase 2 (PDE2) inhibitors(e.g., iso-buto-metho-xanthine (IBMX)), phosphodiesterase 3 (PDE3)inhibitors, phosphodiesterase 4 (PDE4) inhibitors (e.g., rolipram,HT0712), etc.) (see also, e.g., U.S. Pat. No. 6,458,829B1; U.S.Publication No. 2002/0028842A1 (published Mar. 7, 2002)); and modulatorsof protein kinases and protein phosphatases, which mediate CREB proteinactivation and CREB-dependent gene expression. Augmenting agents can beexogenous CREB, CREB analogs, CREB-like molecules, biologically activeCREB fragments, CREB fusion proteins, nucleic acid sequences encodingexogenous CREB, CREB analogs, CREB-like molecules, biologically activeCREB fragments or CREB fusion proteins.

Augmenting agents can also be CREB function modulators, or nucleic acidsequences encoding CREB function modulators. CREB function modulators,as used herein, have the ability to modulate CREB pathway function. By“modulate” is meant the ability to change (increase or decrease) oralter CREB pathway function.

Augmenting agents can be compounds which are capable of enhancing CREBfunction in the CNS. Such compounds include, but are not limited to,compounds which affect membrane stability and fluidity and specificimmunostimulation. In a particular embodiment, the augmenting agent iscapable of transiently enhancing CREB pathway function in the CNS.

CREB analogs, or derivatives, are defined herein as proteins havingamino acid sequences analogous to endogenous CREB. Analogous amino acidsequences are defined herein to mean amino acid sequences withsufficient identity of amino acid sequence of endogenous CREB to possessthe biological activity of endogenous CREB, but with one or more“silent” changes in the amino acid sequence. CREB analogs includemammalian CREM, mammalian ATF-1 and other CREB/CREM/ATF-1 subfamilymembers.

CREB-like molecule, as the term is used herein, refers to a proteinwhich functionally resembles (mimics) CREB. CREB-like molecules need nothave amino acid sequences analogous to endogenous CREB.

Biologically active polypeptide fragments of CREB can include only apart of the full-length amino acid sequence of CREB, yet possessbiological activity. Such fragments can be produced by carboxyl or aminoterminal deletions, as well as internal deletions.

Fusion proteins comprise a CREB protein as described herein, referred toas a first moiety, linked to a second moiety not occurring in the CREBprotein. The second moiety can be a single amino acid, peptide orpolypeptide or other organic moiety, such as a carbohydrate, a lipid oran inorganic molecule.

Nucleic acid sequences are defined herein as heteropolymers of nucleicacid molecules. The nucleic acid molecules can be double stranded orsingle stranded and can be a deoxyribonucleotide (DNA) molecule, such ascDNA or genomic DNA, or a ribonucleotide (RNA) molecule. As such, thenucleic acid sequence can, for example, include one or more exons, withor without, as appropriate, introns, as well as one or more suitablecontrol sequences. In one example, the nucleic acid molecule contains asingle open reading frame which encodes a desired nucleic acid product.The nucleic acid sequence is “operably linked” to a suitable promoter.

A nucleic acid sequence encoding a desired CREB protein, CREB analog(including CREM, ATF-1), CREB-like molecule, biologically active CREBfragment, CREB fusion protein or CREB function modulator can be isolatedfrom nature, modified from native sequences or manufactured de novo, asdescribed in, for example, Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, New York (1998); and Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold SpringHarbor University Press, New York. (1989). Nucleic acids can be isolatedand fused together by methods known in the art, such as exploiting andmanufacturing compatible cloning or restriction sites.

Typically, the nucleic acid sequence will be a gene which encodes thedesired CREB protein, CREB analog, CREB-like molecule, CREB fusionprotein or CREB function modulator. Such a gene is typically operablylinked to suitable control sequences capable of effecting the expressionof the CREB protein or CREB function modulator, preferably in the CNS.The term “operably linked”, as used herein, is defined to mean that thegene (or the nucleic acid sequence) is linked to control sequences in amanner which allows expression of the gene (or the nucleic acidsequence). Generally, operably linked means contiguous.

Control sequences include a transcriptional promoter, an optionaloperator sequence to control transcription, a sequence encoding suitablemessenger RNA (mRNA) ribosomal binding sites and sequences which controltermination of transcription and translation. In a particularembodiment, a recombinant gene (or a nucleic acid sequence) encoding aCREB protein, CREB analog, CREB-like molecule, biologically active CREBfragment, CREB fusion protein or CREB function modulator can be placedunder the regulatory control of a promoter which can be induced orrepressed, thereby offering a greater degree of control with respect tothe level of the product.

As used herein, the term “promoter” refers to a sequence of DNA, usuallyupstream (5′) of the coding region of a structural gene, which controlsthe expression of the coding region by providing recognition and bindingsites for RNA polymerase and other factors which may be required forinitiation of transcription. Suitable promoters are well known in theart. Exemplary promoters include the SV40 and human elongation factor(EFI). Other suitable promoters are readily available in the art (see,e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley& Sons, Inc., New York (1998); Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd edition, Cold Spring Harbor University Press, NewYork (1989); and U.S. Pat. No. 5,681,735).

Augmenting agents can enhance CREB pathway function by a variety ofmechanisms. For example, an augmenting agent can affect a signaltransduction pathway which leads to induction of CREB-dependent geneexpression. Induction of CREB-dependent gene expression can be achieved,for example, via up-regulation of positive effectors of CREB functionand/or down-regulation of negative effectors of CREB function. Positiveeffectors of CREB function include adenylate cyclases and CREBactivators. Negative effectors of CREB function include cAMPphosphodiesterase (cAMP PDE) and CREB repressors.

An augmenting agent can enhance CREB pathway function by actingbiochemically upstream of or directly acting on an activator orrepressor form of a CREB protein and/or on a CREB protein containingtranscription complex. For example, CREB pathway function can beaffected by increasing CREB protein levels transcriptionally,post-transcriptionally, or both transcriptionally andpost-transcriptionally; by altering the affinity of CREB protein toother necessary components of the of the transcription complex, such as,for example, to CREB-binding protein (CBP protein); by altering theaffinity of a CREB protein containing transcription complex for DNA CREBresponsive elements in the promoter region; or by inducing eitherpassive or active immunity to CREB protein isoforms. The particularmechanism by which an augmenting agent enhances CREB pathway function isnot critical to the practice of the invention.

Augmenting agents can be administered directly to an animal in a varietyof ways. In a preferred embodiment, augmenting agents are administeredsystemically. Other routes of administration are generally known in theart and include intravenous including infusion and/or bolus injection,intracerebroventricularly, intrathecal, parenteral, mucosal, implant,intraperitoneal, oral, intradermal, transdermal (e.g., in slow releasepolymers), intramuscular, subcutaneous, topical, epidural, etc. routes.Other suitable routes of administration can also be used, for example,to achieve absorption through epithelial or mucocutaneous linings.Particular augmenting agents can also be administered by gene therapy,wherein a DNA molecule encoding a particular therapeutic protein orpeptide is administered to the animal, e.g., via a vector, which causesthe particular protein or peptide to be expressed and secreted attherapeutic levels in vivo.

A vector, as the term is used herein, refers to a nucleic acid vector,e.g., a DNA plasmid, virus or other suitable replicon (e.g., viralvector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g.,adeno-associated viruses), coronavirus, negative strand RNA viruses suchas orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies andvesicular stomatitis virus), paramyxovirus (e.g. measles and Sendai),positive strand RNA viruses such as picornavirus and alphavirus, anddouble stranded DNA viruses including adenovirus, herpesvirus (e.g.,Herpes Simplex virus types 1 and 2, Epstein-Barr virus,cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox and canarypox).Other viruses include Norwalk virus, togavirus, flavivirus, reoviruses,papovavirus, hepadnavirus, and hepatitis virus, for example. Examples ofretroviruses include: avian leukosis-sarcoma, mammalian C-type, B-typeviruses, D-type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin,J. M., Retroviridae: The viruses and their replication, In FundamentalVirology, Third Edition, B. N. Fields, et al., Eds., Lippincott-RavenPublishers, Philadelphia, 1996). Other examples include murine leukemiaviruses, murine sarcoma viruses, mouse mammary tumor virus, bovineleukemia virus, feline leukemia virus, feline sarcoma virus, avianleukemia virus, human T-cell leukemia virus, baboon endogenous virus,Gibbon ape leukemia virus, Mason Pfizer monkey virus, simianimmunodeficiency virus, simian sarcoma virus, Rous sarcoma virus andlentiviruses. Other examples of vectors are described, for example, inMcVey et al., U.S. Pat. No. 5,801,030, the teachings of which areincorporated herein by reference.

A nucleic acid sequence encoding a protein or peptide (e.g., CREBprotein, CREB analog (including CREM, ATF-1), CREB-like molecule,biologically active CREB fragment, CREB fusion protein, CREB functionmodulator) can be inserted into a nucleic acid vector according tomethods generally known in the art (see, e.g., Ausubel et al., Eds.,Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NewYork (1998); Sambrook et al., Eds., Molecular Cloning: A LaboratoryManual, 2nd edition, Cold Spring Harbor University Press, New York(1989)).

The mode of administration is preferably at the location of the targetcells. In a particular embodiment, the mode of administration is toneurons.

Augmenting agents can be administered together with other components ofbiologically active agents, such as pharmaceutically acceptablesurfactants (e.g., glycerides), excipients (e.g., lactose), stabilizers,preservatives, humectants, emollients, antioxidants, carriers, diluentsand vehicles. If desired, certain sweetening, flavoring and/or coloringagents can also be added.

Augmenting agents can be formulated as a solution, suspension, emulsionor lyophilized powder in association with a pharmaceutically acceptableparenteral vehicle. Examples of such vehicles are water, saline,Ringer's solution, isotonic sodium chloride solution, dextrose solution,and 5% human serum albumin. Liposomes and nonaqueous vehicles such asfixed oils can also be used. The vehicle or lyophilized powder cancontain additives that maintain isotonicity (e.g., sodium chloride,mannitol) and chemical stability (e.g., buffers and preservatives). Theformulation can be sterilized by commonly used techniques. Suitablepharmaceutical carriers are described in Remington's PharmaceuticalSciences.

The dosage of augmenting agent administered to an animal is that amountrequired to effect a change in CREB-dependent gene expression,particularly in neurons. The dosage administered to an animal, includingfrequency of administration, will vary depending upon a variety offactors, including pharmacodynamic characteristics of the particularaugmenting agent, mode and route of administration; size, age, sex,health, body weight and diet of the recipient; nature and extent ofsymptoms being treated or nature and extent of the cognitive function(s)being enhanced or modulated, kind of concurrent treatment, frequency oftreatment, and the effect desired.

Augmenting agents can be administered in single or divided doses (e.g.,a series of doses separated by intervals of days, weeks or months), orin a sustained release form, depending upon factors such as nature andextent of symptoms, kind of concurrent treatment and the effect desired.Other therapeutic regimens or agents can be used in conjunction with thepresent invention.

The present invention will now be illustrated by the following example,which is not to be considered limiting in any way.

EXAMPLE

This study was undertaken to demonstrate that a cognitive deficitassociated with mental retardation can be treated with aphosphodiesterase 4 (PDE4) inhibitor in conjunction with a cognitivetraining protocol. The study was conducted using an animal model ofRubinstein-Taybi syndrome (RTS).

RTS is a human genetic disorder characterized by mental retardation andphysical abnormalities including broad thumbs, big and broad toes, shortstature and craniofacial anomalies (Rubinstein, J. H. & Taybi, H., Am.J. Dis. Child., 105:588-608 (1963); Hennekam, R. C. et al., Am. J. Ment.Retard., 96:645-660 (1992); Levitas, A. S. & Reid, C. S., J. Intellect.Disabil. Res., 42(Pt 4):284-292 (1998); and Cantani, A. & Gagliesi, D.,Eur. Rev. Med. Pharmacol. Sci., 2:81-87 (1998)). RTS occurs in about 1in 125,000 births and accounts for as many as 1 in 300 cases ofinstitutionalized mentally retarded people. In many patients, RTS hasbeen mapped to chromosome 16p13.3 (Imaizumi, K. & Kuroki, Y., Am. J.Med. Genet., 38:636-639 (1991); Breuning, M. H. et al., Am. J. Hum.Genet., 52:249-254 (1993); and Masuno, M. et al., Am. J. Med. Genet.,53:352-354 (1994)), the region of the gene encoding the CREB-bindingprotein (CBP) (Petrij, F. et al., Nature, 376:348-351 (1995)). Many RTSpatients are heterozygous for CBP mutations which yield truncations ofthe CBP C-terminus, suggesting that a dominant-negative mechanism maycontribute to the clinical symptoms (Petrij, F. et al., Am. J. Med.Genet., 92:47-52 (2000)).

Mice carrying a truncated form of CBP show several developmentalabnormalities similar to patients with RTS. RTS patients suffer frommental retardation, while long-term memory formation is defective inmutant CBP mice. A critical role for cAMP signaling duringCREB-dependent long-term memory formation appears to be evolutionarilyconserved.

Methods

Mice

Generation of CBP^(+/−) mice was described by Oike et al. (HumanMolecular Genetics, 8:387-396 (1999)). CBP^(+/−) mice are an acceptedmouse model of Rubinstein-Taybi syndrome, particularly because (i) themolecular lesion (truncated protein) in CBP^(+/−) mice is similar tothose known for some RTS patients, (ii) CBP function in CBP^(+/−)heterozygous mice is reduced but not blocked and (iii) long-term memoryformation, but not learning or short-term memory, appear specifically tobe disrupted in these mutant animals (Oike, Y. et al., Human MolecularGenetics, 8:387-396 (1999)). For these studies, animals were generatedby crossing CBP^(+/−) mice to C57BL/6 females (Jackson laboratory). Themice were genotyped with a PCR protocol as described previously (Oike,Y. et al., Human Molecular Genetics, 8:387-396 (1999)). Age- (12 to 14weeks old by the time of handling) and gender-matched mutant mice andwildtype littermates were used for all experiments.

The mice were kept on 12:12 light-dark cycle, and the experiments wereconducted during the light phase of the cycle. With the exception oftraining and testing times, the mice had ad lib access of food andwater. The experiments were conducted according to Animal WelfareAssurance #A3280-01 and animals were maintained in accordance with theAnimal Welfare Act and the Department of Health and Human Service guide.

Object Recognition Training and Testing

Mice were handled for 3-5 minutes for 5 days. The day before training,an individual mouse was placed into a training apparatus (a Plexiglasbox of L=48 cm; W=38 cm and H=20 cm, located into dimly-illuminatedroom) and allowed to habituate to an environment for 15 minutes (seealso Pittenger, C. et al., Neuron, 34:447-462 (2002)). Training wasinitiated twenty-four hours after habituation. A mouse was placed backinto the training box, which contained two identical objects (e.g. asmall conus-shape object), and allowed to explore these objects. Theobjects were placed into the central area of the box and the spatialposition of objects (left-right sides) was counterbalanced betweensubjects. Among experiments, training times varied from 3.5 to 20minutes.

Three separate, and otherwise experimentally naïve, sets of animals wereused. The first set was used for experiments summarized in FIGS. 3 and 4(n=10 per genotype). The second set was used for the experimentsummarized in FIG. 5 (wildtype mice, n=20). The third set was used forthe experiment summarized in FIG. 6 (n=8 per genotype). For eachexperiment, the same set of animals was used repeatedly with different(new) sets of objects for each repetition. All experiments were designedand performed in a balanced fashion, meaning that: (i) for eachexperimental condition (e.g. a specific dose-effect and/orgenotype-memory effect) 2-6 experimental mice and 2-6 control mice wereused; (ii) experiments with HT0712 injections consisted of thevehicle-injected mice and mice injected with 2-3 different doses ofHT0712; (iii) each experimental condition was replicated 2-4 independenttimes, and replicate days were added to generate final number ofsubjects.

Five-to-eight sessions were performed on each set of mice. Each mousewas trained and tested no more than once per week and with a one-weekinterval between testing. In experiments with drug-injections (seebelow), vehicle-injected mice and high/low-dose-injected mice, werecounterbalanced. In each experiment, the experimenter was unaware(blind) to the treatment of the subjects during training and testing.

To test for memory retention, mice were observed for 10 minutes 3 and 24hours after training. Mice were presented with two objects, one of whichwas used during training, and thus was ‘familiar’ and the other of whichwas novel (e.g. a small pyramid-shape object). The test objects weredivided into ten sets of two “training” plus on “testing” objects, and anew set of objects was used for each training session. After eachexperimental subject, the apparatus and the objects were cleaned with90% ethanol, dried and ventilated for a few minutes.

Drug Compound Administration

Twenty minutes before training, mice were injected in their home cageswith the indicated doses of HT0712((3S,5S)-5-(3-cyclopentyloxy-4-methoxy-phenyl)-3-(3-methyl-benzyl)-piperidin-2-one;also known as IPL 455,903)), Rolipram (in 1% DMSO/PBS) or with vehiclealone (1% DMSO/PBS). HT0712 has the following formula:

wherein “Me” means “methyl” and “cPent” means “cyclopentyl”. HT0712 canbe prepared using the methodology provided in U.S. Pat. No. 6,458,829B1,the teachings of which are incorporated herein by reference.

HT0712 was administered intraperitoneally (i.p.) at doses: 0.001 mg/kg;0.005 mg/kg; 0.01 mg/kg; 0.05 mg/kg; 0.1 mg/kg, 0.15 mg/kg and 0.2mg/kg. Rolipram (Sigma) was administered i.p. at dose 0.1 mg/kg. Drugcompounds were injected with one week interval to allow sufficientwash-out time (the half-life for HT0712 and Rolipram <3 hours). Inaddition, vehicle- and drug-injected mice were counterbalanced fromexperiment to experiment. Such design allowed at least two weekswash-out time between repeated usages of high doses. Nodose-accumulating effects were observed with repeated injectionsbetween/within the groups.

Data Analysis

The experiments were videotaped via an overhead video camera system.Types were reviewed by a blinded observer and the following behavioralparameters were determined: time of exploration of an each object; thetotal time of exploration of the objects; number of approaches to theobjects; and time (latency) to first approach to an object. Thediscrimination index was determined as described previously (Ennaceur,A. & Aggleton, J. P., Behav. Brain Res., 88:181-193 (1997)). The datawere analyzed by Student's unpaired t test using statistical softwarepackage (Statwiew 5.0.1; SAS Institute, Inc). All values in the text andfigure legends are expressed as mean±SEM.

Results

CBP is a transcriptional co-activator that binds to phosphorylated CREB(cAMP-response element binding protein) transcription factor to regulategene expression (Lonze, B. E. & Ginty, D. D., Neuron, 35:605-623(2002)). CREB-dependent gene expression has been shown to underlielong-term memory formation in several vertebrate and invertebratespecies (Poser, S. & Storm, D. R., Int. J. Dev. Neurosci., 19:387-394(2001); Bailey, C. H. et al., Nat. Rev. Neurosci., 1:11-20 (2000);Dubnau, J. & Tully, T., Ann. Rev. Neurosci., 21:407-444 (1998); andMenzel, R., Learn Mem., 8:53-62 (2001)), leading to the intriguingspeculation that mental redardation in RTS patients may derive fromreduced CBP function during long-term memory formation (D'Arcangelo, G.& Curran, T., Nature, 376:292-293 (1995)). To this end, Oike et al.(Human Molecular Genetics, 8:387-396 (1999)) generated a C-terminaltruncation mutation in mouse CBP, which appears to act in adominant-negative fashion to recapitulate many of the abnormalitiesobserved in RTS patients. Homozygous CBP^(+/−) mutants are embryoniclethal, while heterozygous CBP^(+/−) mice show reduced viability, growthretardation, retarded osseous maturation and hypoplastic maxilla (Oike,Y. et al., Human Molecular Genetics, 8:387-396 (1999)). Importantly,CBP^(+/−) mice showed normal learning and short-term memory butdefective long-term memory for two passive avoidance tasks,substantiating the notion that normal CBP function is required formemory formation (Oike, Y. et al., Human Molecular Genetics, 8:387-396(1999)).

A high-throughput drug screen was accomplished using human neuroblastomacells, which were stably transfected with a luciferase reporter genedriven by a CRE (cAMP response element) promoter (a drug screen forenhancers of CREB function) (Scott, R. et al., J. Mol. Neurosci.,19:171-177 (2002)). Cells were exposed to drug for two hours and thenstimulated with a suboptimal dose of forskolin for another four hours.Compounds were selected that had no effect on their own but thatsignificantly increased forskolin-induced CRE-luciferase expression.Among the dozens of confirmed hits for several molecular targetsidentified from this screen, inhibitors of PDE4 were numerous.

As described herein, the PDE4 inhibitors HT0712 and rolipram, which hasbeen shown previously to affect performance in animal models of memory(Barad, M. et al., Proc. Natl. Acad. Sci. USA, 95:15020-15025 (1998);and Vitolo, O. V. et al., Proc. Natl. Acad. Sci. USA, 99:13217-13221(2002)), both produced robust effects on CRE-luciferase expression andon expression of a CRE-dependent endogenous gene, somatostatin (FIG. 2).Other PDE4 inhibitors are expected to produce similar effects.

Initial experiments on normal, young adult mice established thatlong-term memory formation after contextual fear conditioning wasenhanced by PDE4 inhibitors (e.g., HT0712 and rolipram), delivered (i)directly to the hippocampus, (ii) intraventricularly or (iii)intraperitoneally (Scott, R. et al., J. Mol. Neurosci., 19:171-177(2002)). Specifically, these drugs enhanced memory formation by reducingthe amount of training required to produce maximal long-term memory.

To determine whether these drugs could ameliorate memory defects causedby molecular lesions in the CREB pathway, the mouse model ofRubinstein-Taybi syndrome was used particularly because (i) themolecular lesion (truncated protein) in mice was similar to those knownfor some RTS patients, (ii) CBP function in CBP^(+/−) heterozygous micewas reduced but not blocked and (iii) long-term memory formation, butnot learning or short-term memory, appeared specifically to be disruptedin these mutant animals (Oike, Y. et al., Human Molecular Genetics,8:387-396 (1999)).

Long-term memory defects in CBP^(+/−) mutants have been reported onlyfor fear-based tasks (Oike, Y. et al., Human Molecular Genetics,8:387-396 (1999)). Hence, it was first determined if CBP^(+/−) mutantmice also had defective long-term memory for a different type of task.Object recognition is a non-aversive task which relies on a mouse'snatural exploratory behavior. During training for this task, mice arepresented with two identical novel objects, which they explore for sometime by orienting toward, sniffing and crawling over. Mice then willremember having explored that object. To test for such memory, mice arepresented at a later time with two different objects, one of which waspresented previously during training and thus is “familiar,” and theother of which is novel. If the mouse remembers the familiar object, itspends more time exploring the novel object. By analogy to an objectrecognition-based ‘non-matching to sample’ task in monkeys and rats(Mishkin, M., Nature, 273:297-298 (1978); Mishkin, M. & Appenzeller, T.,Sci. Am., 256:80-89 (1987); and Wood, E. R. et al., Behav. Neurosci.,107:51-62 (1993)), this task can be performed repeatedly on the sameanimals by exposing them serially to different sets of novel objects.

Initially, CBP^(+/−) mutants and their wildtype (normal) littermateswere given 15 minutes to explore a novel object during training and thentheir memory retention was tested three and 24 hours later (FIG. 3).Three-hour memory appeared normal, but 24-hour memory was significantlyreduced, in CBP^(+/−) mutants. These results indicate that CBP^(+/−)mutant mice have impaired long-term memory, but normal short-termmemory, for object recognition. These findings extend the observationsof Oike et al. (Human Molecular Genetics, 8:387-396 (1999)) to anethologically relevant, non-aversive behavior and confirm the notionthat loss-of-function mutations in CBP can yield specific defects inlong-term memory formation.

To evaluate the PDE4 inhibitors, drug or vehicle alone were administeredi.p. to normal mice and CBP^(+/−) mutants 20 minutes before a 15-minutetraining session (FIG. 4). As in the previous experiment, 24-hour memoryretention was significantly reduced in CBP^(+/−) mutants in the absenceof drug. In striking contrast, however, a single administration of 0.10mg/kg PDE4 inhibitor (e.g., HT0712 or rolipram) restored 24-hour memoryin CBP^(+/−) mutants to normal levels.

To address whether the drugs' effects were specific to the molecularlesion in CBP, the training protocol was changed and dose sensitivitycurves were determined for mutant and wild-type animals. The 15-minutetraining protocol produces maximum 24-hour retention in the wildtypemice used here. Consequently, drug-induced memory enhancement inwildtype mice was not observed (FIG. 4). By reducing training to a3.5-minute protocol, 24-hour retention was near zero in wildtype mice,thereby allowing an evaluation of the enhancing effects of the PDE4inhibitors. Because CBP^(+/−) mutants had less functional CBP thanwildtype animals, a higher concentration of drug may be required in themutants than in wildtype mice to produce equivalent levels of memoryenhancement. In essence, the molecular lesion in CBP would act to shiftthe dose sensitivity for a PDE4 inhibitor to enhance memory formation.

Initially the dose-response curve for wildtype mice was quantified (FIG.5). In mice treated with vehicle alone, the 3.5-minute training protocoldid not produce any appreciable 24-hour memory. At concentrations below0.05 mg/kg or at 0.50 mg/kg, HT0712 failed to produce any memoryenhancement. Twenty-four hour memory retention was significantlyincreased, however, at concentrations of 0.05, 0.10, and 0.15 mg/kg forHT0712. Next, memory retention was compared between CBP^(+/−) mutantsand wildtype animals at selected concentrations of HT0712 (FIG. 6). Theinitial effective dose was found to differ between mutant and wildtypeanimals. At a dose of 0.05 mg/kg for HT0712, wildtype animals showedsignificant enhancement of 24-hour memory, but CBP^(+/−) mutants didnot. Memory enhancement was first seen in CBP^(+/−) mutants at the nexthigher dose of HT0712 (0.10 mg/kg). Similarly, the peak effective doseappears shifted to a higher concentration in mutants (0.15 mg/kg) thanin wildtype mice (0.10 mg/kg).

It was also considered whether HT0712 might be increasing performance inthe task nonspecifically by affecting perception of the training context(objects) or the motivation to explore objects during training ortesting. The latency to first approach an object during training, thetotal number of approaches to an object and the total exploration timewere analyzed. In all experiments, no differences between genotypesand/or drug treatments were observed in the latency to first approach.CBP^(+/−) mutant mice showed increases in total exploration time and inthe total number of object-approaches, but drug treatments did notchange these measures, and these behavioral responses were notcorrelated with Discrimination Indices.

The data herein indicate that the memory impairments observed forCBP^(+/−) mutants in an object recognition task can be ameliorated byinhibitors of PDE4. These PDE inhibitors likely enhance signaling toCREB/CBP during memory formation by increasing cAMP levels in responseto experience-dependent changes in neural activity (Barad, M. et al.,Proc. Natl. Acad. Sci. USA, 95:15020-15025 (1998); and Nagakura, A. etal., Neuroscience, 113:519-528 (2002)). Given the molecular andpathological similarities between these CBP^(+/−) mice and patients withRubinstein-Taybi syndrome, the findings herein indicate that PDE4inhibitors represent an effective therapy for the mental retardationassociated with this heritable condition by rescuing a functional defectin long-term memory formation and rendering the patient capable ofbenefitting from cognitive training and experience. The findings hereinalso imply that PDE4 inhibitors represent an effective therapy for othermental retardation syndromes, including Angelman syndrome,neurofibromatosis, Coffin-Lowry syndrome, down syndrome, Rett syndrome,myotonic dystrophy, fragile X syndrome (e.g., fragile X-1, fragile X-2)and William's syndrome, by treating a cognitive dysfunction or cognitivedeficit associated with the mental retardation and rendering the patientcapable of benefitting from cognitive training and experience.

All publications, patent and patent applications mentioned in thisspecification are incorporated herein by reference to the same extent asif each individual publication, patent or patent application wasspecifically and individually incorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed:
 1. A method comprising: (a) providing cognitive training to an animal during rehabilitation of said animal from a trauma-dependent loss of cognitive function under conditions sufficient to produce an improvement in performance by said animal of a cognitive task whose deficit is associated with said trauma-dependent loss of cognitive function; (b) administering to said animal in conjunction with said cognitive training an inhibitor of phosphodiesterase 2; (c) repeating said providing and said administering of steps (a) and (b) one or more times; and (d) producing a long-lasting improvement in performance of said task relative to the improvement in performance of said task produced by cognitive training alone; wherein the inhibitor of phosphodiesterase 2 is an augmenting agent which enhances CREB pathway function; and wherein said trauma-dependent loss of cognitive function is associated with a cerebrovascular disease, brain trauma, or head injury.
 2. A method comprising: (a) providing cognitive training to an animal during rehabilitation of said animal from a trauma-dependent loss of cognitive function under conditions sufficient to produce an improvement in performance by said animal of a cognitive task whose deficit is associated with said trauma-dependent loss of cognitive function; (b) administering to said animal in conjunction with said cognitive training an inhibitor of phosphodiesterase 2; (c) repeating said providing and said administering of steps (a) and (b) one or more times; and (d) reducing the number of training sessions sufficient to produce said improvement in performance relative to the improvement in performance produced by cognitive training alone; wherein the inhibitor of phosphodiesterase 2 is an augmenting agent which enhances CREB pathway function; and wherein said trauma-dependent loss of cognitive function is associated with a cerebrovascular disease, brain trauma, or head injury.
 3. The method of claim 1, wherein said trauma-dependent loss of cognitive function is associated with a cerebrovascular disease, and said cerebrovascular disease is selected from the group consisting of stroke and ischemia.
 4. The method of claim 2, wherein said trauma-dependent loss of cognitive function is associated with a cerebrovascular disease, and said cerebrovascular disease is selected from the group consisting of stroke and ischemia.
 5. The method of claim 1, wherein said trauma-dependent loss of cognitive function is associated with a brain trauma, and said brain trauma is selected from the group consisting of subdural hematoma and brain tumor.
 6. The method of claim 2, wherein said trauma-dependent loss of cognitive function is associated with a brain trauma, and said brain trauma is selected from the group consisting of subdural hematoma and brain tumor.
 7. The method of claim 1, wherein said animal is a mammal.
 8. The method of claim 7, wherein said mammal is a human.
 9. The method of claim 2, wherein said animal is a mammal.
 10. The method of claim 9, wherein said mammal is a human.
 11. The method of claim 1, wherein said cognitive training comprises spaced training sessions.
 12. The method of claim 2, wherein said cognitive training comprises spaced training sessions.
 13. The method of claim 3, wherein said cognitive training comprises spaced training sessions.
 14. The method of claim 4, wherein said cognitive training comprises spaced training sessions.
 15. The method of claim 8, wherein said cognitive training comprises spaced training sessions.
 16. The method of claim 10, wherein said cognitive training comprises spaced training sessions.
 17. The method of claim 11, wherein said inhibitor of phosphodiesterase 2 is administered before one or more training sessions.
 18. The method of claim 12, wherein said inhibitor of phosphodiesterase 2 is administered before one or more training sessions.
 19. The method of claim 13, wherein said inhibitor of phosphodiesterase 2 is administered before one or more training sessions.
 20. The method of claim 14, wherein said inhibitor of phosphodiesterase 2 is administered before one or more training sessions. 